DESCRIPTION (from the applicant's abstract): The catalytic function of beta-lactamases is the primary mechanism of bacterial resistance to beta-lactam antibiotics. A multidisciplinary approach has been outlined for the study of beta-lactamases, which builds on the mechanistic findings from the principal investigator's laboratory in the current cycle of funding. Four Specific Aims are outlined. Specific Aim 1 details the plans for cloning, expression, large-scale production, crystallization and characterization of the mechanism of action of the oxo-10 beta-lactamase from Pseudomonas aeruginosa. This enzyme is a prototypic member of the class D of beta-lactamases, which are understood the least currently. This enzyme is the parent enzyme for a number of expanded-spectrum beta-lactamases, which have been identified clinically recently. Specific Aim 2 describes the plans for investigation of the inhibitor-resistant TEM ("IRT") beta-lactamases. A subset of beta-lactamases is being identified since 1992 that resist inhibition by clinically used inhibitors. Four of these enzymes carry individually single mutations of mechanistic consequence for the inhibitor-resistant trait. These enzymes will be investigated in mechanistic and crystallographic efforts to elucidate their catalytic properties. Specific Aim 3 proposes a series of experiments in development of inhibitors for beta-lactamases that inhibit more than one class of these enzymes. Specific Aim 4 details research in elucidating the mechanistic and structural aspects for turnover of imipenem, a clinically important beta-lactam antibiotic, by the common class A TEM-1 beta-lactamase. This system exhibits certain desirable characteristics that make it amenable to structural analysis at every step of the catalytic process for turnover. These efforts collectively will help define the dynamic system of random mutation in the resistance genes and the process of selection that have given considerable diversity to these resistance enzymes in clinical settings.